Alzheimer's Disease
Alzheimer's disease is an increasingly prevalent form of neurodegeneration that accounts for approximately 50-60% of the overall cases of dementia among people over 65 years of age. Pathologically, Alzheimer's disease neurodegeneration is characterised by prominent atrophy of corticolimbic structures with neuronal death and loss of neuronal synapses, neurofibrillary tangle (NFT) formation, and the formation of senile plaques containing deposits of amyloid β1-42 (Aβ42) aggregates in the brain [Francis P T 1999]. The duration of the progressive cognitive decline is approximately 7 years from the occurrence of first signs until death. It is assumed that the clinical phase is preceded by a 15-30 years preclinical period of continuous deposition of amyloid plaques and neurofibrillary tangles. Age of onset and progression of the disease are largely determined by causative gene mutations and by genetic susceptibility factors. Several environmental risk factors may add to the individual genetic risk factors. Genetic factors known to be involved in the familial form of Alzheimer's disease with early onset of the disease are: mutations in presenilin 1 (PS1), presenilin 2 (PS2), and amyloid precursor protein (APP) genes, and the presence of the apolipoprotein E4 allele. However, the majority (95%) of Alzheimer's disease cases is sporadic and heterogeneous.
Currently, clinical diagnosis of Alzheimer's disease can only be established at later stages of the disease, when cognitive perfotniance is significantly decreased and paralleled by structural alterations of the brain. The clinical diagnostic work up requires a careful medical history; physical and neurological examination; blood, urine and cerebrospinal fluid (CSF) examinations to exclude metabolic and medical disease states that might masquerade Alzheimer's disease; detailed psychometric examinations to assess mental status and cognitive performance, and imaging techniques such as computed tomographic scan or magnetic resonance imaging of the brain. Diagnostic evaluations at expert centres reach an accuracy of about 80-85%. Due to the fact that these tests are expensive and time consuming, and are particularly inconvenient to patients, there is an increasing need for easy-accessible specific diagnostic biomolecule markers, which can be measured in body fluids, such as CSF, blood or urine, and which have a high positive predictive value for diagnosis of Alzheimer's disease, or would help to distinguish Alzheimer's disease from other forms of dementia. Furthermore, reliable markers sensitive to disease progression may constitute surrogate parameters, a major prerequisite for the evaluation and development of new causal oriented and disease modifying therapeutic strategies in Alzheimer's disease.
Since CSF directly surrounds the brain, changes in its protein composition may most accurately reflect pathologic conditions that are associated with specific alterations of the protein expression patterns. Over the last decade, a number of biological abnormalities have been reported in the cerebrospinal fluid (CSF) of Alzheimer's disease patients, in particular altered levels of the Aβ1-42 fragment of the amyloid precursor protein, and altered levels of the hyperphosphorylated tau protein. The sensitivity and specificity of these markers, however, is low or only modest [The Ronald and Nancy Reagan Research Institue of the Alzheimer's Association and the National Institute on Aging Working Group, 1998, Robles A 1998, Termissen C E et al., 2002].
Hence, there is a need for novel biomarkers with sufficient sensitivity and specificity for (i) detecting Alzheimer's disease as early as possible, and (ii) to allow disease differentiation from other types of dementia or neurodegenerative diseases, and (iii) monitoring therapeutic efficacy as surrogate parameter, e.g. in clinical drug development, and to initiate pharmacotherapy as early as possible and postpone loss of memory and disease progression.
Protein Chip Technology
A Protein chip technology called Surface Enhanced Laser Desorption/Ionisation time of flight mass spectrometry (SELDI-TOF MS) has recently been developed to facilitate protein profiling of complex biological mixtures [Davies H A 2000, Fung E T 2001, Merchant M 2000].
Protein chip mass spectrometry has already been used by several groups to detect potentially novel biomarkers of prostate and bladder [Adam B L 2001] or breast cancer [Wulfkuhle J D 2001] in serum, seminal plasma, nipple fluid, urine or cell extracts. For a review on biomarker search using SELDI-TOF MS, see [Issaq H J 2002].
Cystatin C
Initially described in 1961 in cerebrospinal fluid (CSF), cystatin C (γ trace or post-γ globulin, Acc. No. P01034) is a small cystein proteinase inhibitor present in all human body fluids at physiologically relevant concentrations. The physiological role of cystatin C is likely to regulate extracellular cysteine protease activity, which results from microbial invasion or release of lysosomal proteinases from dying or diseased cells. Cystatin C colocalises with β-amyloid (Aβ) within the arteriolar walls in Alzheimer's disease brains and cerebral amyloid angiopathy [Levy E 2001]. There are two common haplotypes of the CST3 gene coding for cystatin C (A and B) that differ from each other at three sites: two single base pair changes in the promoter region and one in the signal peptide domain that causes an amino acid substitution (alanine to threonine). Recently, case control studies found associations of CST3 with increased risk for late onset Alzheimer's disease [Crawford F C 2000, Finckh U 2000, Beyer K 2001].
Hereditary cerebral hemorrhage with amyloidosis, Icelandic type (HCHWA-I), also called hereditary cystatin C amyloid angiopathy (HCCAA), is an autosomal dominant form of cerebral amyloid angiopathy (CAA). The amyloid deposited in the brain vessel's walls is composed mainly of a variant of cystatin C characterised by the presence of the Leu68-Gln substitution [Cohen 1983, Ghiso 1986]. This pathology is also coupled to a decreased concentration of this major cystein proteinase inhibitor in cerebrospinal fluid and leads to its amyloid deposition in the brain [Grubb A O 1984].
Leung-Tack et al have also purified two N-terminal truncated isoforms of cystatin C in urine from one patient who had received renal transplant. According to their data, (des1-4) cystatin C has an inhibiting effect on two functions of human peripheral mononuclear cells (PMN): O2− release and phagocytosis, which may be due to the N-terminal sequence ‘KPPR’. Their data support a potentially important role for cystatin C as a possible immunomodulator during inflammation. Accumulating evidence indicates that increased free radical mediated damage to cellular function contributes to the ageing process and age-related neurodegenerative disorders. Oxidative stress may play a role in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS). Although free-radical damage to neurons may not be the primary event initiating these diseases, it appears that free-radical damage is involved in the pathogenetic cascade of these disorders.
Beta-2-Microglobulin
Beta-2-microglobulin (Ace. No. P01884) constitutes the small constant component of the class I major histocompatibility complex (CMH) and its presence in biological fluids represents the balance between membrane protein turnover and elimination. Since this peptide seems to be increased in some diseases characterised by an elevation of the immune response, its quantification in body fluids has become a useful index of immunological state in vivo [Hoekman et al 1985]. The function of this protein is unclear, but it seems to be implicated in diseases, which involve glial cell destruction [Ernrudh et al 1987].
The technical problem which is solved by the present invention is the provision of improved methods for diagnosing Alzheimer's disease and/or monitoring the progression of Alzheimer's disease in a subject.
Neurosecretary Protein (VGF)
VGF (human VGF, Acc.-No.: O15240) is a secretory peptide precursor that is expressed and processed by neuronal cells [Canu et al. 1997]. In situ hybridization studies in the adult rat central nervous system have revealed that the VGF mRNA is widely distributed throughout the brain with prominent expression in the hippocampus, entorhinal cortex, and neocortex. Furthermore, it has been shown that VGF transcription and secretion is selectively upregulated by neurotrophins like NGF and BDNF, and by depolarization in vitro. Increased BDNF expression can be observed in dentate gyrus and CA3 regions of the hippocampus, which are tissues that appear to die early in Alzheimer Disease pathogenesis.